We have discovered a novel mechanism (or mechanisms) of resistance to ?-lactams that is independent of the two known mechanisms of staphylococcal resistance to beta-lactams: pencillinase and PBP2a. This type of resistance was identified during experiments in which a methicillin-susceptible S. aureus strain was passaged in the presence of a so-called fifth generation anti-MRSA cephalosporin, ceftobiprole. Whole genome sequencing of a resistant mutant revealed mutations in genes encoding the following proteins: PBP4, a non-essential, low-molecular weight penicillin-binding protein; GdpP, a putative signaling protein; and AcrB, a putative transporter. We hypothesize that mutations in one or more of these genes confer high-level ?-lactam resistance. To test this hypothesis, three specific aims are proposed. Aim 1: To determine which mutations in pbp4 confer high-level ?-lactam resistance. pbp4 missense mutations will be repaired using a standard allelic replacement mutagenesis protocol; repaired strains will be compared to the parent for changes in ?- lactam resistance. Alternatively, mutations will be introduced into susceptible S. aureus strains to determine the effect on susceptibility. ?-lactam antibiotic binding and enzymatic functions of PBP4 will also be assayed. Aim 2: To determine the role of gdpP in mediating susceptibility and resistance to ?-lactam antibiotics. Mutation in gdpP is associated with increased expression of pbp4 and with resistance to ?-lactams. The genetic approach described above will be used to determine whether GdpP regulates expression of pbp4 and whether GdpP impacts resistance by pbp4 or other mechanisms. Aim 3: To identify other non-mecA genes that mediate ?-lactam antibiotic resistance. Besides pbp4 and gdpP, a missense mutation was identified in arcB of the resistant mutant. Its contribution to resistance is not known. Allelic replacement experiments will be performed to determine whether AcrB regulates pbp4 expression or influences resistance phenotype. Whole genome resequencing of another ceftobiprole passage mutant, SRB, which also has pbp4 mutations but no mutations in gdpP or acrB, will be performed to identify other mutations that contribute to resistance. The overall goal of the proposed research is to define the molecular basis for mecA-independent resistance to ?-lactams, which could lead to a better understanding of ?-lactam antibiotic effects and identification of novel drug targets.